Source: Christopher P. Corbo¹, Jonathan F. Blaize¹, Elizabeth Suter^1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301

Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed "extremophiles", these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.

Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.

Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.

Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).

Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.

One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.

This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

1. Preparation

1. Before beginning, wash hands thoroughly and put on appropriately sized gloves.
2. Sterilize work surface with 5% sodium hypochlorite (bleach) and dry thoroughly.
3. Place an inoculating loop in an empty 120 mL Erlenmeyer flask so that it does not touch the bench top while working.

2. Growth Media and Cultures

1. Gather four plates of Mannitol Salt Agar (MSA), Eosin Methylene blue agar (EMB), and eight tr

Mannitol Salt Agar (MSA): This medium is selective for gram positive organisms that are able to survive in 6.5% sodium chloride. The gram-negative organisms Escherichia coli and Proteus vulgaris should not be able to grow on this medium because of the high salt concentration. S. epidermidis and S. aureus should be able to grow. The media is differential between the two because the S. aureus is able to ferment the mannitol - tur

Different bacterial species are able to grow in different environments and are able to use different carbon sources as a way of generating energy. When working with these as cultures in the lab, it is important to know the components of the growth media being worked with and to match the growth media to the bacterial species. Scientists and diagnosticians can also exploit the varying biochemical reactions as a way to isolate different species from others and as a way to distinguish and identify bacteria in a mixed enviro

1. Fernandez, L. A. Exploring prokaryotic diversity: there are other molecular worlds. Molecular Microbiology, 55 (1), 5-15 (2005).
2. Grattieri, M., Suvira, M., Hasan, K., & Minteer, S. D. Halotolerant extremophile bacteria from the Great Salt Lake for recycling pollutants in microbial fuel cells. Journal of Power Sources, 356, 310-318 (2017).
3. Wendt-Potthoff K. & Koschorreck, M. Functional Groups and Activities of Bacteria in a Highly Acidic Volcanic Mountain Stream and Lake in Patagonia, Argentina. Microbial Ecology, 1, 92 (2002).
4. Lee, L. S., Goh, K. M., Chan, C. S., Annie Tan, G. Y., Yin, W.-F., Chong, C. S., & Chan, K.-G. Microbial diversity of thermophiles with biomass deconstruction potential in a foliage-rich hot spring. Microbiology Open, 7 (6), e00615 (2018)
5. Ito, T., Sekizuka, T., Kishi, N., Yamashita, A., & Kuroda, M. Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria. Gut Microbes, 10 (1), 77-91. (2019)
6. Vartoukian, S. R., Palmer, R. M., & Wade, W. G. Strategies for culture of "unculturable" bacteria. FEMS Microbiology Letters, 309 (1), 1-7. (2010)
7. Harris, T. M., Rumaseb, A., Beissbarth, J., Barzi, F., Leach, A. J., & Smith-Vaughan, H. C. Culture of non-typeable Haemophilus influenzae from the nasopharynx: Not all media are equal. Journal of Microbiological Methods, 137, 3-5. (2017)
8. Wang, Y.-Y., Ai, P., Hu, C.-X., & Zhang, Y.-L. Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose. International Journal of Hydrogen Energy, 36 (1), 390-396. (2011)
9. Pascual, A., Basco, L. K., Baret, E., Amalvict, R., Travers, D., Rogier, C., & Pradines, B. Use of the atmospheric generators for capnophilic bacteria Genbag-CO₂ for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malaria Journal, 10, 8 (2011).
10. Summanen, P., McTeague, M., Väisänen, M.-L., Strong, C., & Finegold, S. Comparison of Recovery of Anaerobic Bacteria Using the Anoxomat®, Anaerobic Chamber, and GasPak®Jar Systems. Anaerobe, 5, 5-9. (1999)
11. de la Fuente-Núñez, C., Reffuveille, F., Fernández, L., & Hancock, R. E. Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies. Current Opinion in Microbiology, 16, 580-589. (2013)
12. Possé, B., De Zutter, L., Heyndrickx, M., & Herman, L. Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol-positive and -negative O157. FEMS Microbiology Letters, 282 (1), 124-131. (2008)